This invention relates to treatment of diseases characterized by production of cell surface markers using antibody-targeted compositions. More particularly, this invention relates to chimeric organisms that express an antibody fragment and to the use of such chimeric organisms in treatment of diseases characterized by production of cell surface markers.
Many recent gene therapy approaches have exploited the specificity of antibody binding to target cancer cell lines in order to deliver either drugs or immune responses to an actual tumor location. Most cancer cell lines misregulate cell surface proteins and polysaccharides, and are thus easily distinguished from normal somal cells by antibodies (R. E. Hawkins et al., Gene Therapy (1998), 5:1581-1583). It is apparent that established carcinomas have successfully avoided activating the immune response within their hosts. Direct attempts to rectify this by recruiting the body""s humoral immune response to tumors by injection of murine derived antibodies can unfortunately cause serious and even life threatening human anti-mouse responses (R. K. Jain et al., J Natl. Cancer Inst. (1989) 81:570-576 and D. Colcher et al., J. Nat. Cancer Inst. (1990) 82:1191-1197). In addition, the overall penetration of antibodies into tumors is limited due to the high molecular weights of these molecules (K. A. Chester et al., Adv. Drug Delivery Rev. (1996) 22:303-313).
In an attempt to limit both the size of the antibody and the mouse-character of the antibody, single chain antibodies (scFvs) that encapsulate the binding features of the Fv region of the antibody without the bulk of the native antibody sequence in the c1, c2, and c3 domains have been developed. One methodology to generate scFvs involves tethering the antigen binding domains of VH and VL together using a short flexible peptide linker (R. E. Bird et al., Science (1988) 242:423-426). Another approach involves the generation de novo of molecular diversity, instead of generating monoclonal antibodies in mice. By using combinatorial antibody libraries on the surface of filamentous bacteriophage screened against immobilized antigen, a single polypeptide chain that is amenable to fusion with other proteins can be generated (J. S. Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; J. McCafferty, Nature (1990) 348:552-554; R. H. J. Begent et al., Nature Med. (1996) 2:979-984, reviewed in K. A. Chester et al., Adv. Drug Delivery Rev. (1996) 22, 303-313). The scFvs obtained by either methodology above show better tumor penetration, but therapeutic application is still in early stages (G. Reitmuller et al., Lancet (1994) 343: 1177-1183). However, fusions between imaging agents and scFvs have found wide acceptance and extensive application in tumor imaging and radiochemotherapeutic delivery (see J. Bhatia et al., Cancer (1999) 85:571-577 and A. M. Wu et al., Tumor Targeting (1999) 4:47-58 and references therein).
Antibody recognition has also been used to target cancer cells by incorporation of an scFv into the envelope protein of a retrovirus (S. J. Russell et al., Nuc. Acids Res. (1993) 21:1081-1085 and F. Martin et al., Human Gene Therapy (1998) 9:737-746). This targeting is modest, but offers some promise, as has been demonstrated for certain types of melanoma (Martin 1998). In addition, adenovirus infection has been used to allow the transient expression of tumor-targeting scFv fusion proteins in whole organisms with moderate success (H. A. Whittington et al., Gene Therapy (1998) 5:770-777). Unfortunately, low survivability of adenoviruses carrying antibody generating expression vectors limits their impact.
The most promising therapeutic techniques relying on the specificity of antibody binding focus on engineering T-cells that express antibody fragments fused to surface proteins, and are thus directed to tumor surfaces (recent work reviewed in F. Paillard, Human Gene Therapy (1999) 10:151-153). Some of these T-cells are at present in clinical trials. Strategies used to date, however, have drawbacks, including limited efficacy against established tumors, though demonstrating some slowing of tumor metastasis (R. P. McGuinness et al, Human Gene Therapy (1999) 10:165-173). Limited effectiveness against established tumors may be due to the inability of the T-cells to penetrate solid cell masses (Paillard 1999). True protection against establishment of invasive carcinoma was obtained only by coinjection of modified T-cells with the tumorogenic line. In clinical applications, this may permit stabilization and localization of established tumors, but not reductive treatment. Another potential problem is that suicide signals T-cells use to induce apoptosis, like tumor necrosis factor I, are often not functional against carcinomas. Even when they are effective, successful cancer cell lines will rapidly adapt to apoptotic signals, and have even been known to induce apoptosis in attacking T-cells (K. Shiraki, Proc. Natl. Acad. Sci. USA (1996) 94:6420-6425). In addition, T-cells bearing these chimeras are assembled separately for each patient ex vivo due to possible MHC incompatibilities that could result in serious allergic reactions were T-cells from other humans introduced therapeutically.
Candida albicans is the most commonly isolated invasive fungal pathogen in humans. This organism is representative of several that switch between two major classes of morphology. The first morphology is the ellipsoid blastospore. Like most yeast, C. albicans assumes this architecture when growing non-pathogenically. Upon binding of C. albicans to mammalian tissues (i.e. via the I domain of the INT-1 protein), the cell morphology switches to various filamentous forms, including germ tubes and hyphae, that are capable of aggressively invading host tissue (reviewed by R. A. Calderone, Microbol. Rev. (1991) 55, 1-20). Systemic infection of a vulnerable host by C. albicans results in high levels of mortality. For example, more than 30% of immunocompromised HIV patients are systemically infected despite appropriate treatment regimes. In addition, C. albicans infection commonly leads to death in premature infants, diabetics, and surgical patients. To date, the ability of this pathogenic organism to infect cells when the cell morphology switches to a filamentous form has not been utilized for therapeutic purposes, such as in cancer therapy.
Thus, the need exists in the art for new and better compositions and methods of their use for treating various types of cancers and other diseases associated with production of an abnormal protein.
The present invention overcomes these and other problems in the art by providing chimeric organisms having a chimeric surface integrin-like protein in which the I domain has been replaced by an antibody fragment that binds a disease-associated antigen on a cell. Binding of the antibody fragment to the disease-associated antigen on the cell triggers virulent transformation of the chimeric pathogenic organism and allows the organism to infect the cell.
In one embodiment according to the present invention, there are provided chimeric pathogenic C. albicans modified to contain an integrin1 (INT1) fusion protein in which the I domain is replaced by an antibody fragment that binds to a disease-associated antigen on a diseased cell. The chimeric C. albicans further contains a disabled wild-type high affininity iron transporter (CAFTR) gene, and a DNA construct comprising a wild-type CAFTR gene under the control of an enhanced filamentous growth protein (EFG1p) response element, wherein binding of the antibody to the disease-associated antigen triggers expression of the CAFTR gene in the DNA construct and filamentous transformation in the chimeric pathogenic C. albicans. 
In another embodiment according to the present invention, there are provided methods for treating a disease associated with the presence of cells having a disease-associated surface antigen in a subject in need thereof by administering to the subject a therapeutically effective amount of an invention chimeric pathogenic organism so as to cause binding of the antibody fragment to the disease-associated antigen on the cells, thereby treating the disease by triggering infiltration of the chimeric pathogenic C. Albicans into the cells without substantial damage to healthy cells.
In yet another embodiment, the present invention provides methods for generating a chimeric therapeutic organism from a pathogenic organism that possesses in the wild-type an integrin-like protein with an I domain. In the invention methods, the I domain in the integrin-like protein of the pathogenic organism is replaced with an antibody fragment that binds to a disease-associated antigen on a diseased cell. In the chimeric therapeutic organism, virulent transformation occurs upon binding of the antibody fragment to the disease-associated antigen on the cell.